RecQ helicase-catalyzed DNA unwinding detected by fluorescence resonance energy transfer

被引：12
作者：
Zhang, XD
Dou, SX
Xie, P
Wang, PY [1 ]
Xi, XG
机构：
[1] Chinese Acad Sci, Inst Phys, Beijing Natl Lab Condensed Matter Phys, Lab Soft Matter Phys, Beijing 100080, Peoples R China
[2] Ecole Normale Super, CNRS, UMR 8113, Lab Biotechnol & Pharmacol Genet Appl, F-94235 Cachan, France
来源：
ACTA BIOCHIMICA ET BIOPHYSICA SINICA | 2005年 / 37卷 / 09期
关键词：
helicase; DNA unwinding; kinetics; stopped-flow; fluorescence resonance energy transfer (FRET);
D O I：
10.1111/j.1745-7270.2005.00084.x
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
A fluorometric assay was used to study the DNA unwinding kinetics induced by Escherichia coli RecQ helicase. This assay was based on fluorescence resonance energy transfer and carried out on stopped-flow, in which DNA unwinding was monitored by fluorescence emission enhancement of fluorescein resulting from helicase-catalyzed DNA unwinding. By this method, we determined the DNA unwinding rate of RecQ at different enzyme concentrations. We also studied the dependences of DNA unwinding magnitude and rate on magnesium ion concentration. We showed that this method could be used to determine the polarity of DNA unwinding. This assay should greatly facilitate further study of the mechanism for RecQ-catalyzed DNA unwinding.
引用
收藏
页码：593 / 600
页数：8
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